Environmental Pollution 145 (2007) 171e178

www.elsevier.com/locate/envpol

The effect of winter stress on Ilex aquifolium L.previously

fumigated with ozone
Jonathan Ranford*, Kevin Reiling
Applied Sciences, Staffordshire University, College Road, Stoke-on-Trent, Staffordshire, ST4 2DE, UK
Received 2 September 2005; received in revised form 16 March 2006; accepted 21 March 2006

Exposure to ozone decreases the winter hardening capability and markedly increases
the frost sensitivity of Ilex aquifolium.

Abstract

European Holly (Ilex aquifolium) received either charcoal-filtered air (CFA) or CFA with 70 nl l1 ozone added for 7 h day1 over a 28 day
period. Plants were then transferred into cooling incubators for hardening (4  C day/2  C night; day length 12 h) for 7 days and then to the
frosting stage (2  C day and 5, 10 or 15  C night) for 4 days. The plants were then placed in ambient conditions. Treatment produced
significant differences in chlorophyll fluorescence data. Stomatal conductance was significantly higher for the ozone treatments though both
showed a general decline over all temperature regimes. Ozone also significantly increased electrolyte leakage and reduced winter survival. These
results show that ambient concentrations of ozone can reduce the tolerance of I. aquifolium to freezing stress, which may have serious impli-
cations for its establishment and survival.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Ozone; Ilex aquifolium; Frost stress; Chlorophyll fluorescence; Electrolyte leakage; Stomatal conductance

1. Introduction Ozone exposure in European northern latitudes often leads

Recent works dealing with frost, climate change and ozone
impacts upon vegetation (Inouye, 2000; Ashmore, 2005;
Broadmeadow and Ray, 2005) suggest that altered timing of
frost episodes may have far-reaching impacts on communities
and ecosystems. In over-wintering plants, survival of frost and
desiccation depends on optimum morphological development,
accumulation of adequate reserves during the summer and on
hardening in the autumn (Barnes et al., 1988). A reduction in
reserves or perturbations of metabolism and timing of frosts
could reduce the effectiveness of winter hardening. In
response to ozone there is the potential that energy normally
needed for growth and the development of frost hardiness is
instead utilised for maintenance purposes and to repair dam-
age (Bäck et al., 1993).
* Corresponding author. Tel.: þ44 782 294892; fax: þ44 782 294986.
E-mail address: jon.ranford@staffs.ac.uk (J. Ranford).
to plants becoming more susceptible to injury than in southern
areas. In these areas short summer nights do not give enough
time for the repair process (De Temmerman et al., 1992) and
there is a higher ozone flux inside the leaves due to a lower
vapour pressure deficit (Karlsson et al., 2004). Northern areas
are also subjected to greater temperature variation and impor-
tantly an increasing number of frosting episodes are predicted
(NETGAP, 2001). Development of a winter hardening state is
an important feature of northern perennial species because it
aids survival against frost (Taulavuori et al., 1997). Frost resis-
tance is conferred through changes in cytosolic compounds
and in membrane structure, which enables cells to withstand
the stress of extracellular ice formation (Larcher, 1995). The
effects of ozone on winter stress have been previously investi-
gated in several native plant species with several important
modifications documented. Waite et al. (1994) reported
decreased cold tolerance on Picea rubens Sarg. seedlings
fumigated with various concentrations of ozone; Foot et al.

0269-7491/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2006.03.019
172 J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 171e178
(1997) reported increased frost sensitivity following long-term
fumigation of Calluna vulgaris L. and Prozherina et al. (2003)
indicated detrimental impacts of springtime frosts and early
growth on Betula pendula Roth. Assessments on crop species,
although fewer because of the number of winter crop species
have also shown damaging impacts (von Tiedemann and
Firsching, 2000; Khan and Soja, 2003).
An important experimental tool in assessing plant stress
physiology often used to measure the effect of ozone exposure
is chlorophyll fluorescence (Reiling and Davison, 1994; Potter
et al., 1996; Gimeno et al., 1999; Bortier et al., 2000; Bortier
et al., 2001; Zheng et al., 2002; Ishii et al., 2004) and is often
specifically used in assessing cold-induced stress (Fisker et al.,
1995; Filiault and Stier, 1999; Burr et al., 2001). Many exper-
iments have demonstrated that these changes have a fundamen-
tal impact on plant metabolism often being linked to a decline
in photosynthetic rates and thus productivity (Heath, 1996;
Potter et al., 1996; Gimeno et al., 1999; Grams et al., 1999).
The reduction in photosynthesis after exposure to ozone has
been attributed to several factors including the decrease in sto-
matal conductance, the decline in the quantity and activity of
Rubisco and the capacity of electron transport (Clark et al.,
1996; Kull et al., 1996; Kellomaki and Wang, 1997; Reichena-
uer et al., 1997; Bortier et al., 2000).
This study investigated ozone impacts on reduction in win-
ter tolerance by looking at physical changes within the leaves
compared with whole plant effects of leaf loss and production
reported in an earlier experiment (Ranford and Reiling, in press).
In contrast to the typical response to ozone, Ilex aquifolium is
one of the few species that has an increase in stomatal conduc-
tance (Ranford and Reiling, in press). This may have perturba-
tions regarding the water balance of the plant with winter
desiccation being a serious problem for evergreen plants
(Leverenz et al., 1999; Soleimani et al., 2003). In addition
weak root pressure caused by ozone-induced freezing injury
could represent a risk of shoot dieback and tree decline as suf-
ficient root pressure is required for springtime refilling of xylem
embolisms caused by winter cavitation of vessels and to ensure
an adequate foliar water supply (Zhu et al., 2002).
2. Methods
2.1. Plant growth conditions and fumigation
First year I. aquifolium plants obtained from a commercial supplier (Kerry
Hill Nurseries, Staffordshire, UK) were potted into square 10 cm pots (John
Innes no. 3) and placed in a glasshouse for 28 days in June 1999. The plants
were then transferred to four controlled environment chambers (AAF Ltd
Cramlington, Northumberland, UK), 125 in each, and left to acclimatise to
chamber conditions for 14 days. All chambers were ventilated with char-
coal-filtered air (CFA). The environmental conditions in each of the chambers
were uniform and no significant between-chamber variation was detected.
The mean temperature in the chambers was 18  C  2  C day, 12  C  2  C
night and illumination was provided by metal halide lamps (Siemens Son-T
Harrier 250 W unit fitted with Osram Powerstar HQI-T 250 W/D lamp) pro-
viding a photon flux density of 550 mmol m2 s1 at plant canopy height over
a 16 h photoperiod.
All experimental chambers were ventilated to receive CFA 24 h d1
(<5 nl l1 O3) with a mean air speed of w1 m s1 at plant height. In addition
half of the chambers received a supplement of ozone resulting in a level of
70 nl l1 O3  4 nl l1 O3 from 10.00 to 17.00 (AOT40 5880) for a period
of 28 days. Ozone concentration was measured continuously using a Dasibi
R1000 ozone monitor (Dasibi Environmental Corp, Glendale, California,
USA). Ozone was generated by an ozonater (Wallace and Tiernan, Tonbridge,
Kent, UK) from charcoal scrubbed air. To prevent contamination by higher ox-
ides of nitrogen (Brown and Roberts, 1988) the ozone enriched air, post fumigation
was passed through water prior to injection into the main chamber inlet.
The fumigation period started on June 29 and terminated on July 26, 1999.
After fumigation the plants were returned to the greenhouse for 28 days for
post-chamber adaptation.
2.2. Frost stress
Post fumigation, 40 plants of each treatment were transferred into six
Sanyo MIR 252 cooling incubators (Sanyo Electric Co, Japan) and the subse-
quent experimental procedure is detailed in Fig. 1. This comprised of an initial
hardening process of 4  C day/2  C night, with a day length of 12 h, for 7 days
and then a frosting period, at 2  C day and 5, 10, or 15  C night for 4
days. The incubators were programmed to fall at 2  C h1 to the minimum
temperature, hold for 4 h, then to rise to 2  C. There were two cooling incu-
bators per frosting temperature. Post-frosting plants were subjected to a read-
justment period for 7 days of 4  C day/2  C night before being placed into
ambient conditions (Staffordshire University, Stoke-on-Trent, Staffordshire,
UK) for the remainder of the experiment.
Chlorophyll fluorescence and stomatal conductance were measured prior
to removal of leaves for measurement of electrolyte leakage. Once leaves
were removed, the plants were removed from the experiment in case the
leaf removal itself imparted a measurable stress.
2.3. Plant measurements
Chlorophyll fluorescence was recorded daily at standardised time (10 am),
using a Photosynthetic Efficiency Analyser Mk II fluorescence system (Hansa-
tech Instruments, King’s Lynn, Norfolk, UK). The adaxial surfaces of 10 fully
expanded leaves of varying age per plant were randomly selected from 30
plants per treatment and fast fluorescence kinetics was recorded after
a 30 min dark adaptation period. A dark adaptation phase is needed to reverse
all non-photochemical quenching fluorescence (Krause and Weis, 1991) before
fluorescence can be measured. In the dark-adapted state, initial fluorescence
(F0), when all PSII reaction centres are open can be determined. Maximal fluo-
rescence (Fm), when all PSII reaction centres are closed is then assessed with
saturating light. Maximum quantum yield for electron transport by open PSII
centres (photochemical efficiency) was calculated as Fv:Fm ¼ (Fm  F0)/Fm.
In addition to being a general indication of stress (Lichtenthaler, 1996) the
Fv/Fm ratio is correlated with the quantum yield of net CO2 assimilation
(Bjorkman and Deeming, 1987).
Stomatal conductance was recorded daily at a standardised time (2 pm)
using an AP4 portable porometer (Delta-T Devices, Cambridge, Cambridge-
shire, UK) during the frosting experiment. Ten fully expanded leaves per plant
were randomly selected from 30 plants per treatment and gs recorded per frost-
ing temperature.
After non-destructive parameters were recorded, three fully expanded
leaves per plant were removed (10 plants per treatment) and three leaf discs,
6 mm in diameter, were sampled. These plants were then no longer used in the
experiment in case leaf removal imparted stress and therefore influenced fur-
ther experimental results. The leaf discs were then placed into a test tube with
18 cm3 of water and shaken for 8 h in a water bath held at 4  C. The elec-
trical conductivity of the bathing solution was measured, then the total electro-
lyte content of the discs was recorded after maceration. Leakage was
expressed as a percentage of the total electrolyte content. Angular transforma-
tion was performed on the data before statistical analysis. This method has
been adapted from Barnes et al. (1988).
2.4. Winter survival
After the frost treatments the plants were placed into ambient conditions
and then monitored for winter survival. Simple counts (n) of living and dead
 J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 171e178 173

CFA

125 125 125 125

14 d

CFA CFA + 70 nl-1O3

125 125 28 d 125 125

Greenhouse 28 d Post-chamber adaptation

Plant Measurements for Hardening and Frosting.

Hardening 40 40 40 40 40 40
o o 1. C.F. 30 plants, (10 leaves per plant) per treatment
4 C day/2 C night
7d 40 40 40 40 40 40 2. S.C. 30 plants, (10 leaves per plant) per treatment
3. E.L. 10 plants, (3 leaves, 5 discs) per treatment
After E.L. Plants removed from frosting experiment

-5oC -10oC -15oC -5oC -10oC -15oC

1 night 35 35 35 35 35 35 - 60 plants
35 35 35 35 35 35
F
R
O 2 nights 25 25 25 25 25 25 - 60 plants
S 25 25 25 25 25 25
T
I
N 3 nights 20 20 20 20 20 20 - 60 plants
G 20 20 20 20 20 20
- 60 plants
15 15 15 15 15 15
4 nights 15 15 15 15 15 15

Hardening
All plants 4°C day/2°C night7 d

Winter Survival
Randomised block design of plants consisting of the above 30 plants per
frosting/fumigation treatment. Total 180 plants

Winter Survival Assessment February 24th

Key

Controlled Cooling C.F = Chlorophyll fluorescence

Environmental n Incubators n1 E.L. = Electrolyte Leafage
Chambers n2 n = number of plants, 1– CFA. 2 - CFA + 70 nl-1O3

Fig. 1. Method flow diagram.

174 J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 171e178

plants were recorded on March 24, 1999. Survival was expressed as a 0.820
percentage of the number of living plants to the original number of
plants. a*
0.800

Mean Fv:Fm
0.780 b*
2.5. Statistical analysis
Control -5
Statistical analyses were performed using the SPSS statistical package 0.760 Ozone -5 b*
(SPSS Inc., Chicago, Illinois, USA) to assess the effect of ozone fumigation. Control -10
0.740 Ozone -10
Hardening data were subject to ANOVA; time-course data were subject to two-
Control -15
way repeated measures (RM) ANOVA; two-way nested ANOVA was applied Ozone -15
between hardening data and the fourth nightly measurement. Where appropri- 0.720
ate post hoc LSD was applied. H 1 2 3 4
Nights of Frosting

Fig. 3. The effect on Fv:Fm of ozone fumigation (70 nl1 28 d 7 h d1 June 29,
3. Results 1999) prior to an artificial frosting regime on I. aquifolium. H ¼ hardening,
ozone (open symbols) or control maintained in charcoal-filtered air (closed
3.1. Chlorophyll fluorescence symbol). a ¼ ANOVA, b ¼ two-way nested ANOVA. Significant differences
from the control are denoted. *P < 0.05.

Ozone fumigated plants exhibited a significant increase in

were no significant differences within the nightly measure-
F0 for the hardening treatment (Fig. 2) compared with control.
ments within each treatment.
In addition subsequent frosting treatment produced a signifi-
cant increase in F0 for the ozone-treated plants. The magnitude
3.3. Electrolyte leakage
of F0 perturbation was linked to the magnitude of the frosting
temperature. There were no significant differences within the
Leaves from ozone-treated plants exhibited a large increase in
nightly measurements within each treatment.
leakage of w30% from hardening to 5  C, with another simi-
The magnitudes of the Fv and Fm both showed a general
lar increase after the 10 and 15  C treatments (see Fig. 5).
increase with ozone while the Fv/Fm ratio showed an overall
Treatments that were previously fumigated with ozone were
reduction from hardening and continued decreases over the
significantly higher for all treatments except the 15  C. There
four nights of frosting (Fig. 3) with all but the 15  C treat-
were no significant differences within the nightly measurements
ment being significantly different with ozone. There were no
within each treatment.
significant differences within the nightly measurements within
each treatment.
3.4. Winter survival

3.2. Stomatal conductance
Ozone-treated plants showed a significant increase com-
pared with controls for all treatments (Fig. 4). An overall reduc-
tion in gs linked to the magnitude of the frosting temperature,
the largest reductions being at lowest temperatures. There
Plants that received supplementary ozone before the frosting
episode had a marked decrease in survival (see Fig. 6). The
5  C frosting treatment reduced survival of ozone
fumigated plants by w35%, the 10  C treatment
by w20% and the 15  C treatment by w5%. Climatological
and AOT40 data for this period are presented in Table 1.

0.09 200
180
0.08 b* a*
160
mean gs mmol-2 s-1

140
0.07
b*
Mean Fo

120
0.06 b*
100
b* 80 b*
0.05 b*
60
a*
40
0.04
20
0.03 0
H -5 -10 -15 H -5 -10 -15
Temperature °C Temperature °C

Fig. 2. The effect on F0 of ozone fumigation (70 nl1 28 d 7 h d1 June 29,
1999) prior to an artificial frosting regime on I. aquifolium. H ¼ hardening,
ozone (open symbols) or control maintained in charcoal-filtered air (closed
symbol). a ¼ ANOVA, b ¼ two-way nested ANOVA. Significant differences
from the control are denoted. *P < 0.05.
Fig. 4. The effect on stomatal conductance of ozone fumigation (70 nl1 28 d
7 h d1 June 29, 1999) prior to an artificial frosting regime on I. aquifolium.
H ¼ hardening, ozone (open symbols) or control maintained in charcoal-fil-
tered air (closed symbol). a ¼ ANOVA, b ¼ two-way nested ANOVA. Signif-
icant differences from the control are denoted.*P < 0.05.
 J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 171e178 175

70 Table 1
Summary of the AOT40a and climateb data for the winter survival period,
60 Stoke-on-Trent, Staffordshire
% Electrolyte Leakage

50 b* Date AOT40 Temperature (  C) Frost Precip. Sunshine

Mean Mean (d) (mm) (h)
40
max. min.
30 September 1999 1411 19.6 10.0 1 125.3 178.5
b**
October 244 14.3 6.3 3 80.8 116.4
20
November 453 10.6 4.1 10 34.2 66.1
10 December 1666 7.8 1.1 13 79.8 44.8
a* January 2000 6031 8.0 1.4 7 23.0 59.8
0 February 5172 9.5 1.9 11 55.1 102.2
H -5 -10 -15
Temperature °C Total 17 720 n/a n/a 45.0 398.2 567.6
a
1 Air Quality Archive (primary data).
Fig. 5. The effect on electrolyte leakage of ozone fumigation (70 nl 28 d b
The Met Office.
7 h d1 June 29, 1999) prior to an artificial frosting regime of I. aquifolium.
H ¼ hardening, ozone (open symbols) or control maintained in charcoal-fil-
tered air (closed symbol). a ¼ ANOVA, b ¼ two-way nested ANOVA. Signif-
icant differences from the control are denoted. *P < 0.05; **P < 0.01. The chlorophyll fluorescence data exhibited clear signifi-
cant differences for hardening and frosting. The hardening
process shows a significant decrease in Fv/Fm, which is also
4. Discussion evident in the frosting treatments with the largest falls being
associated with the coldest temperatures. Of all the fluores-
The negative impact of exposure to ozone on frost tolerance cence parameters, Fv/Fm continued to fall on subsequent
on I. aquifolium is mostly consistent with that reported in the frosting nights indicating increasing levels of stress. The
published literature for other species. Waite et al. (1994), post-hardening fall indicates that the plants were stressed
showed that P. rubens seedlings treated with ozone (50 nl l1) even before they received the frosting treatment with possible
had significantly less cold tolerance and a study on C. vulgaris photoinhibitory reductions in Fv/Fm occurring (Maxwell and
demonstrated that naturally occurring frost exposure resulted in Johnson, 2002) with ozone showing a significant impact on
increased damage and mortality of ozone-treated (70 nl l1) the process. Ozone has previously been shown to delay the
plants (Foot et al., 1997). Tests with spruce seedlings and rate at which plants attain winter hardiness and freezing resis-
low temperatures suggested that plant vitality during winter tance after hardening (Lucas et al., 1988; Edwards et al.,
may be impaired by the ozone concentration (Braun and 1990). The fluorescence data also indicate a threshold effect
Fluckiger, 1995). Ozone has been shown to predispose spruce between 5 and 10  C with ozone influencing the position
to frost injury by delaying the rate at which they attain winter of this threshold. This study supports the idea that interactions
hardiness during the autumn (Lucas et al., 1988). However, between ozone and climatic stress, in particular, drought and
ozone enrichment on the Vaccinium vitis-idaea L. had no mea- frost hardiness, can result in potentially detrimental effects
surable effect on frost hardiness (Taulavuori et al., 2001). to a range of European trees (Skarby et al., 1998).
It is believed that injury by ozone is generally the conse-
90 quence of an inability to repair or compensate for altered
80
membrane permeability (Wellburn et al., 1994). The damage
to the membrane is reflected by the increased loss of electro-
70
lytes for the ozone-treated plants. This study indicated that
60 membranes are more permeable and have increased cellular
% Survival

50
sensitivity to frost when exposed to ozone. Similar findings
have been reported on C. vulgaris (Foot et al., 1996, 1997).
40
Increasing F0 in ozone-treated plants may indicate sensitivity
30 at the cellular level particularly the PSII reaction centres or as-
20 sociated pigment protein complexes (Calatayud and Barreno,
2001). However, F0 also increases if the optical properties
10
are altered by leaf water content changes. The observed
0 increase in stomatal conductance in ozone plants could explain
-5 -10 -15
Frosting Temperature °C
this. Increase in F0 is thought to reflect a subsequent change of
structural nature and reduction in photochemistry (Krause and
Fig. 6. The effect of ozone fumigation (70 nl1 28 d 7 h d1 June 29, 1996) Weis, 1991; Maxwell and Johnson, 2002). The repair of
prior to an artificial frosting regime (four nights of 5, 10 or 15  C) on structural damage will require plant resources and take
the winter survival of I. aquifolium (March 24th, 2006), previously exposed
to 4 nights of artificial frost. Ozone (open bars) or control maintained in char- a certain period to be accomplished. This may be reflected
coal-filtered air (closed bars). Winter survival rates expressed as a percentage in the long-term effects of ozone observed on I. aquifolium
of the original number of plants. (Ranford and Reiling, in press).
176 J. Ranford, K. Reiling / Environmental Pollution 145 (2007) 171e178
The increased electrolyte leakage for ozone-treated plants
combined with the fluorescence data suggests a membrane
dysfunction. This is a potentially serious problem because
membrane integrity is crucial for withstanding the damage as-
sociated with freezing. The plasma membrane is a primary
target for ozone damage (Skarby et al., 1998) with cell mem-
branes becoming more permeable and hence increases cellular
sensitivity to frost (Foot et al., 1996). Ozone has been shown
to damage cell membranes by changing the lipid composition
of the plasmalemma (Barnes and Davison, 1992) with most
studies showing fatty acid unsaturation/saturation ratios de-
creasing after exposure to ozone (Wellburn and Wellburn,
1994; Sandelius et al., 1995; Anttonen and Karenlampi,
1996). A reduction in fatty acid unsaturation by ozone can re-
strict the ability of the cell to accommodate internal freezing
(Skarby et al., 1998). However, a study on I. aquifolium ob-
served that the desaturation of the fatty acids in thylakoid
membranes during the hardening process did not correlate
with alterations in frost resistance (Rutten and Santarius,
1988). Instead the increase in cold hardiness was closely re-
lated to an increase in cellular sap concentration, mainly by
sucrose accumulation when the minimum air temperature
dropped to 0  C or below. The degree of unsaturation of polar
lipids of the thylakoids may contribute to maintaining optimal
functional efficiency of the membranes at low temperatures
rather than to avoiding freezing damage (Rutten and Santar-
ius, 1988). With the observed significant increase in F0 and
Fv for ozone-treated plants, ozone may affect both these pro-
cesses resulting in the reduction in cold hardiness and frost
tolerance.
Ozone exposure reduces both the capacity for C fixation
and use of fixed C (carbon allocation) (Andersen, 2003; Fiscus
et al., 2005) and hence ozone will reduce the availability of
C-based cryoprotectants (Skarby et al., 1998); the decreased
photochemical efficiency shown by the chlorophyll data may
indicate a similar problem in this study.
The significant increase in gs in I. aquifolium is an atypical
response to ozone but has been reported in an earlier study
(Ranford and Reiling, in press). This reduced water-use effi-
ciency may be a particular problem when water is unavailable
and therefore imposes an additional drought effect (winter des-
iccation). Studies have shown that stomatal conductance may
rise or fall in response to ozone damage, dependent on geno-
type, ozone concentration and sensitivity of the guard cells to
ozone compared with surrounding epidermal cells (Robinson
et al., 1998). The higher stomatal conductance and decreased
Fv in ozone-treated plants may indicate that the guard cells of
I. aquifolium have become damaged. Previous studies have
shown that ozone has exacerbated winter damage by winter
desiccation and/or photoinhibition rather than a change in
frost tolerance (Barnes and Davison, 1988; Chappelka et al.,
1990; Mikkelsen and Ropoulsen, 1994; Braun and Fluckiger,
1995).
While the extrapolating of these findings into field situa-
tions on mature trees may be a useful exercise, caution must
be exercised to account for the greater number of variables
that plants experience, e.g. differing light levels, wind,
desiccation and temperature fluctuations. Frosting in the field
may not expose plants to sub-zero temperatures at a constant
rate and exposure to frosting time may be greater than in the
controlled laboratory situation (Foot et al., 1997). Also the
effects of chamber-based experimental conditions and inherent
physiological properties may contribute to increased ozone
sensitivity of tree seedlings (Kolb and Matyssek, 2001;
Manning, 2005). The significance of repeated frost damage
for tree vitality and yield will depend on the proportion of
healthy foliage remaining (Skarby et al., 1998). This study
together with that of Ranford and Reiling (in press) indicates
potential ozone induced problems involving vitality and
productivity for I. aquifolium in native systems.
5. Conclusions
This study demonstrates that exposure to an ambient ozone
concentration interferes with the winter hardening capability
and markedly increases the frost sensitivity and winter survival
of I. aquifolium seedlings. These results supported by the ear-
lier findings of Ranford and Reiling (in press) suggest that in-
creased damage and mortality resulting from ozone exposure
with a carry-over frost interaction may have serious implica-
tions for I. aquifolium, for its establishment and survival.
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